The studies proposed in this MERIT extension applicafion address the molecular mechanisms by which age-induced changes in the microenvironment of Leydig cells, the cells responsible for tesficular androgen biosynthesis, lead to reduced testosterone formation. We propose the following: (i) Changes in the balance between reactive oxygen accumulation and the antioxidant defense system result in increased oxidative stress which, in turn, results in damage to the signal transduction cascade at the plasma membrane and thus in reduced responsiveness of aged Leydig cells to luteinizing hormone (LH). (ii) As a consequence, aged cells produce less cAMP than young cells, resulfing in reduced intracellular cholesterol transport into the mitochondria and therefore reduced substrate availability for testosterone. The first aim is to determine the mechanism by which increased oxidafive stress elicit reducfions in cAMP and testosterone producfion in aged Leydig cells. We will test the hypothesis that in response to increased oxidafive stress, there is defecfive coupling of LH receptors (LHR) to Gs proteins, resulfing in reduced cAMP in response to LH stimulafion. The second aim will test the hypothesis that as a consequence of the blunted ability of LH to sfimulate cAMP producfion in aged Leydig cells, there are reduced levels of translocator protein (TSPO) and steroidogenic acute regualtory protein (STAR)-mobilized cholesterol, and thus less efficient transfer of cholesterol to the inner mitochondrial membrane. We further hypothesize that the reduced expression of TSPO in aged cells results from that reduced expression of Natural Antisense Transcripts (NATs). The third aim is to determine how the age-related changes in Brown Norway rat Leydig cells compare to those in aging human testes. With this aim, we will translate our findings in the rat to the human. To this end, we will measure testosterone and ROS levels in intratesficular fluid from young and aged men, capture Leydig cells from testicular biopsies to analyze steroidogenic enzyme expression and the expression of proteins involved in reactive oxygen producfion and in the anfioxidant defense system, and examine proteins and lipids damaged by changes in the pro/anfioxidant.balance using fixed fissue.